The long-term goal of our research is to understand how Rho GTPase signaling regulates cell migration and invasion. Active cancer cell migration and invasion are critical steps during tumor metastasis, which is the major cause of death in cancer patients. Cell migration and invasion are largely controlled by the actomyosin cytoskeleton that, in many cases, is ultimately modulated by Rho GTPase signaling. As the major components of Rho GTPase signaling, Rho GTPase proteins are primarily activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). There are at least two modes of cell migration: mesenchymal- and amoeboid-mode. The former requires Rac1 activation and matrix proteolysis, while the latter is characterized by membrane blebbing and requires high activity of RhoA. Cancer cells can adopt either mesenchymal- or amoeboid-mode of cell migration yet can switch from mesenchymal- to amoeboid-mode of migration to escape cancer treatments that specifically target matrix proteolysis. Thus, successful cancer treatments require targeting both matrix proteolysis and RhoA/RhoC-signaling. It is evident that a RhoGEF is likely required to promote RhoA/RhoC activation for amoeboid-like cell migration. It is well known that invasive cancer cells can use their membrane blebs to squeeze through the extracellular matrix, thus invading surrounding tissues and eventually leading to tumor metastasis. However, it is largely unknown how RhoGEFs are implicated in the regulation of membrane blebbing and amoeboid-like cell migration. The objective of this proposal is to determine how myosin II- interacting guanine nucleotide exchange factor (MYOGEF) is implicated in the regulation of membrane blebbing and amoeboid-like cell migration as well as to identify the upstream regulatory signals that lead to the activation of MYOGEF. We propose two specific aims. In Aim 1, we will test the hypothesis that MYOGEF is recruited to the bleb membrane, where it stimulates the reassembly of the actomyosin network to initiate the retraction of membrane blebs, thus promoting amoeboid-like cell migration. In Aim 2, we will test the hypothesis that the upstream regulatory signals can activate MYOGEF by disrupting the autoinhibitory intramolecular interactions between the DH domain and the carboxyl-terminal region of MYOGEF.